Chronic sleep disorders are pervasive in society, have poor therapeutic options, and cause an estimated annual economic burden of $100 billion. Despite the impact of sleep disorders, the fact that we sleep for a third of our lives, and the evolutionary conservation of sleep-like states, mechanisms that regulate sleep remain poorly understood. Understanding these mechanisms is important because sleep performs an essential function and is necessary in order to eventually develop therapies for sleep disorders. Progress has been hindered in part by the complexity of mammalian brains and the challenge of performing screens in mammals. To overcome these limitations, we and others recently demonstrated behavioral, anatomical, genetic and pharmacological conservation of sleep between zebrafish and mammals, establishing zebrafish as a simple and inexpensive vertebrate model for sleep. We recently used zebrafish to perform the first large-scale screen for genes that regulate vertebrate sleep. We found that overexpression of the neuropeptide neuromedin U (Nmu) promotes locomotor activity and inhibits sleep. While one study showed that Nmu can transiently disrupt sleep in rats, its role in sleep has not been extensively studied, the mechanism through which it affects sleep is unclear, and a role for nmu-expressing neurons in sleep has not been explored. We will determine the genetic and neurological mechanisms through which Nmu and nmu-expressing neurons regulate sleep using approaches that exploit advantageous features of zebrafish. In Specific Aim 1 we will test the hypothesis that Nmu signaling is required for normal sleep/wake behaviors using nmu and nmu receptor mutants, and explore whether Nmu is required for arousal in specific contexts. We will also determine the genetic mechanisms through which Nmu affects sleep by testing whether zebrafish that lack neuromodulators and neuropeptides known to regulate sleep suppress Nmu gain- and loss-of-function phenotypes. These experiments will identify genetic mechanisms through which Nmu regulates sleep and place it in the context of established sleep pathways. In Specific Aim 2 we will test the hypothesis that nmu-expressing neurons inhibit sleep by using optogenetic and chemogenetic assays to stimulate, inhibit and ablate them, and assay effects on behavior. In Specific Aim 3 we will test the hypothesis that Nmu promotes wakefulness by stimulating corticotropin releasing hormone (crh)-expressing brainstem neurons, and that Crh signaling, the locus coeruleus and noradrenaline are required for Nmu-induced arousal. Validation of this hypothesis will identify a novel neural circuit that regulates arousal. Because disrupted sleep is associated with several neurological disorders and may be causal in some cases, this project may eventually lead to improved therapies for sleep disorders and some neurological disorders. Nmu signaling is a particularly attractive drug target because Nmu acts via G- protein coupled receptors, which are amenable to drug modulation, although much additional work will be needed before the basic research proposed here can be translated into therapies.